核受体相关因子1基因转染神经干细胞移植治疗帕金森病

Transplantation of Nurr1 gene transfected neural stem cells for treatment of Parkinson's disease

目的探讨转染核受体相关因子1(Nurr1)基因的神经干细胞(NSCs)移植至帕金森病(PD)大鼠纹状体后向多巴胺能神经元的分化及对PD大鼠行为的影响。方法应用脑立体定位技术构建单侧PD大鼠模型。将PD大鼠随机分为3组,每组8只。假手术组注射生理盐水,NSCs组移植未转染的NSCs,Nurr1组先将重组质粒载体pEGFP-N1-Nurr1转染NSCs,用RT-PCR方法及免疫荧光染色检测Nurr1基因的表达效果,然后用转染Nurr1基因的NSCs进行移植。细胞用DIL标记后移植至PD大鼠右侧纹状体中,术后2周起用阿朴吗啡诱发旋转试验观测移植后大鼠行为改善情况,12周后用免疫荧光技术检测移植细胞中酪氨酸羟化酶(TH)的表达。结果重组质粒转染后Nurr1基因能在NSCs中过表达。移植后假手术组和NSCs组PD大鼠的旋转圈数均无下降趋势,两者差异无统计学意义(P>0.05);NSCs组移植区可见DIL阳性细胞,但TH免疫阳性细胞较少,平均每视野为(3.21±0.40)个;Nurr1组移植后PD大鼠的旋转圈数从第6周开始下降,移植区DIL/TH双标神经元每视野达(9.28±1.09)个。结论 Nurr1基因过表达能诱导NSCs在PD大鼠纹状体内分化为TH阳性的多巴胺能神经元,并在一定程度上改善大鼠的行为功能。

Objective To study the differentiation of neural stem cells （NSCs） transfected with nuclear receptor related factor 1 （Nurrl） gene into dopaminergic neurons and its behavioral exertion after transplanted into the striatum of Parkinson＇s disease （PD） rats. Methods The unilateral PD rats were prepared by the rat brain stereotaxic technology. The successful PD rats were randomly divided into 3 groups with 8 rats in each group. The rats in the sham group were injected with normal saline. Untransfected NSCs were grafted into the rats in NSCs group. In Nurrlgroup, the recombinant pEGFP-N1-Nurrl was transfected into NSCs first, the expression of Nurrl gene was detected by RT-PCR and immunofluorescence, and then the NSCs were transplanted. Cells were labeled with DIL and transplanted into the right striatum of PD rats. Apomorphine-induced rotation test was performed to observe the behaviour amelioration 2 weeks after transplantation. The expression of tyrosine hydroxylase （TH） was detected by immunofluorescence labeling 12 weeks later. Results Nurrl gene was overexpressed in NSCs after transfected with recombinant plasmid. After transplantation, the rotations of PD rats did not decrease obviously in sham and NSCs groups （P 〉 0. 05）. DIL positive cells were observed in NSCs group, but TH positive neurons were few with （3.21 ±0.40） cells per field. In Nurrl group, the rotations of PD rats began to decrease by the sixth week after transplantation, and the DIL/TH double-labeled neurons reached （9.28±1.09） cells per field in the transplanted area. Conclusion Overexpression of Nurrl may induce the differentiation of NSCs into TH positive dopaminergic neurons in the striatum of PD rats and ameliorate the behavioral function of PD rats.